Methods for monitoring physiological status of a body organ

ABSTRACT

The present invention provides method for monitoring physiological status of an organ in a subject by monitoring morphological changes over time in transplanted tissue on an eye of the subject.

CROSS REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 15/621,058 filed Jun. 13, 2017, which is acontinuation of U.S. patent application Ser. No. 15/278,933 filed Sep.28, 2016, now U.S. Pat. No. 9,702,867 issued Jul. 11, 2017, which is acontinuation of U.S. patent application Ser. No. 13/950,710 filed Jul.25, 2013, now U.S. Pat. No. 9,462,973 issued Oct. 11, 2016, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/676,578,filed Jul. 27, 2012, each incorporated by reference herein in itsentirety.

BACKGROUND

A number of different experimental approaches allow for thequantification of beta cell mass and for eventually displaying theremodeling of islets. In vitro techniques include the analysis ofhistological sections of pancreas by point counting morphometry and thedirect measurements of islet dimensions in dissected pancreatic tissue.Ex vivo imaging techniques comprise the imaging of exteriorized pancreasby confocal microscopy or optical coherence microscopy. Finally, severalnon-invasive in vivo imaging techniques aim at the longitudinalquantification of total beta cell mass, i.e. magnetic resonance imaging(MRI), positron emission tomography (PET), bioluminescence imaging, orcombined multimodal imaging. However, these techniques only provideindirect evidence for islet plasticity, as these various techniques donot offer the possibility to follow morphological changes in individualislets over time.

SUMMARY OF THE INVENTION

The present invention provides methods for monitoring physiologicalstatus of an organ in a subject, comprising monitoring morphologicalchanges over time in transplanted tissue on an eye of the subject,wherein the tissue is from an organ of interest, and wherein themorphological changes over time in the transplanted tissue on the eye ofthe subject indicates a physiological status of the organ of interest inthe subject. In one embodiment, the method is used to monitor efficacyof a course of treatment for a disorder in the subject. In thisembodiment, the course of treatment may comprise administration of atherapeutic to the subject, and wherein the method monitors efficacy ofthe therapeutic in the individual. In a further embodiment, the courseof treatment may comprise a diet and/or an exercise regimen, and themethod monitors efficacy of the diet and/or exercise regimen in theindividual.

In another embodiment the transplanted tissue may be obtained from thesubject. In a further embodiment, the organ of interest may selectedfrom the group consisting of pancreas, lung, heart, brain, kidney,liver, small intestine, large intestine, colon, stomach, gall bladder,esophagus, ureter, urethra, ovary, uterus, breast, spinal cord,prostate, hypothalamus, adrenal gland, pituitary gland, thyroid gland,parathryroid gland, pineal gland, spleen, thymus, rectum, mammary gland,seminal vesicles, glomeruli, fat tissue, tumor, and testes.

In another embodiment, the organ of interest may comprise an exocrinegland, including but not limited to sweat glands, salivary glands,mammary glands, stomach, liver, and pancreas. In another embodiment, theorgan of interest may comprise an endocrine gland, including but notlimited to a pituitary gland, pancreas, ovaries, testes, thyroid gland,and adrenal gland. In one embodiment, the organ of interest is thepancreas. In this embodiment, the tissue may comprise isolatedpancreatic islets of Langerhans.

In various embodiments, the morphological changes may be selected fromthe group consisting of cell size in the tissue, cell volume in thetissue, cell area in the tissue, cell shape in the tissue, cell death inthe tissue, cell proliferation in the tissue, cell mass in the tissue,blood perfusion in the tissue, optical reflectivity of cells in thetissue, and granulation of cells in the tissue. In another embodiment,the morphological changes may be monitored by microscopy. In a furtherembodiment, the subject is a non-human mammal. In one embodiment, thenon-human mammal has an animal model of a human disease. In anotherembodiment, the subject is a human, wherein the human has a diseaserelating to the organ of interest. In various embodiments, the diseasemay comprise diabetes and the organ of interest may be the pancreas, orthe disease may comprise cancer and the organ of interest thus comprisesa tumor. In a further embodiment, the methods may further compriseadministering a therapeutic or test compound to the subject, andmonitoring morphological changes resulting from the administering.

DESCRIPTION OF THE FIGURES

FIG. 1. Increased islet size to adapt to high insulin demand in theob/ob mouse pancreas. (A) representative morphological appearance of theob/ob mouse (left) compared to a control littermate (right), showing astrong obese phenotype; (B) fasted body weight of ob/ob and control miceat different ages shows a rapid increase in body mass in the ob/ob mice;(C) fasted blood glucose and plasma insulin levels in ob/ob and controlmice; (D) image montage of 5 μm-thick sections of ob/ob and (E) controllittermate pancreata, stained by hematoxylin and eosin. Note thestaining of islet sections in light grey; insets in (D, E) show amagnified view of typical islet dimensions and morphology. Mice were (A)4 months, (C) 3 months, and (D, E) 8 months of age. (B, C): values formixed males and females (n=5 males+4 females, and 5 males+5 males forob/ob and control littermates, respectively). Scale=1 mm, insetdimension represents 1 mm×1 mm. Values are average±s.e.m.; **p<0.01;***p<0.001.

FIG. 2. In vivo longitudinal imaging of islet growth. (A) islets from 4weeks old mice were transplanted into the anterior chamber of the eye ofcontrol (upper lane) and ob/ob (lower lane) mice at 4 weeks of age.Photography of transplanted eyes at different time points shows thatindividual islets can be identified and followed longitudinally (seedashed circle); (B) magnified views of islet grafts (framed in A) showthe clearly visible large and tortuous blood vessels in the isletengrafted onto the iris of ob/ob recipient; (C) in vivo imaging ofsingle islets 1 month after transplantation by confocal microscopy showsmorphological differences between islet grafts in control versus ob/obmice. Vascularization is imaged by prior intravenous injection ofFITC-labeled dextran. Note differences in backscatter intensity andvessel diameters; (D) in vivo imaging of islet grafts at different timepoints after transplantation by confocal microscopy; (E) quantificationof islet volumes by analysis of backscatter images reveals asignificantly increased growth in ob/ob (solid lines), as compared tocontrol (dashed lines). Gray lines represent average islet volumes insingle mice, black lines represent averaged values obtained per genotype(n=3); (F) immunohistochemistry analysis shows a strong proliferation ofbeta cells both in ob/ob transplanted eye and pancreas, as seen byinsulin and Ki67 staining; (G) average beta cell area was quantifiedfrom insulin and DAPI staining, demonstrating that beta cells from ob/obmice (solid lines) were significantly larger than those from theircontrol littermates (dashed lines). This hypertrophy was similar andindependent on whether the islets were located in situ in the pancreasor in the transplanted eye (no significant differences). All images arerepresentative. Confocal images are displayed as maximum intensityprojections (MIP) of optical z-stacks. Immunohistochemistry experimentswere performed using 4 months old mice (n≥3 per group). Size bars=100μm. Error bars represent s.e.m.; *p<0.05; **p<0.01.

FIG. 3. Physiological effects of leptin treatment on ob/ob mice. (A)ob/ob mice received daily intraperitoneal injections of leptin between 3and 4 months of age. Body weight, blood glucose and plasma insulinlevels were monitored before, during and after the treatment (beginningand end of treatment are represented by vertical dashed lines); (B)intraperitoneal glucose tolerance tests show impaired glucose handlingin the ob/ob mouse as compared to control littermate at 4 months of age(top traces), which is normalized by leptin treatment but not by shamtreatment (middle traces). The lower bar graph shows area under thecurve (AUC) values from the above traces, demonstrating the beneficialeffect of leptin on glucose handling. Values are average±s.e.m.;**p<0.01; ***p<0.001.

FIG. 4. Leptin treatment reverses dysregulation of ob/ob islets. (A)longitudinal in vivo imaging of islet grafts in ob/ob mice receivingleptin treatment (top) or sham treatment (bottom) between 3 and 4 monthsof age. Vasculature was visualized by tail-vein injection ofdextran-FITC; (B) Islet volume analysis shows a reversal of islet growthduring leptin treatment (dark_bars) as compared to sham treatment (lightbars); there was no difference in islet growth after the end of leptinor sham treatment. (C) immunohistochemistry on transplanted eye andpancreas samples from ob/ob mice at the end of the leptin treatmentdemonstrated the attenuated beta-cell proliferation by immunostainingfor insulin and Ki67 (see FIG. 2F for comparison). Confocal images arerepresentatives and shown as MIPs. Size bars=100 μm. Values areaverage±s.e.m.; ***p<0.001.

FIG. 5. Effect of leptin treatment on intra-islet vascularization.Islets were transplanted into the anterior chamber of the eye of ob/obmice at the age of 4 weeks. At 3 months of age the mice received dailyintraperitoneal injections of leptin. (A) leptin administration had arapid effect on blood vessel diameters, as seen by in vivo imaging ofthe same islet before the beginning of the treatment, and after 1 weekof treatment (vasculature was visualized by tail-vein injection ofdextran-FITC). The same vessel segments could be identified at differenttime points and their diameter measured (see lines). Note that we couldobserve angiogenesis following leptin administration (arrows); (B)longitudinal analysis of individual vessel diameters shows vesselmorphological changes before, during and after leptin treatment (toptraces, 20 vessel segments from islet grafts in ob/ob mouse arerepresented over time, lower bar graph shows corresponding averagemonthly diameter increases). Confocal images are representatives andshown as MIPs. Size bars=100 μm. Values are average±s.e.m.; ***p<0.001;****p<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purification” in Methods inEnzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York,N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides methods for monitoringphysiological status of an organ in a subject, comprising monitoringmorphological changes over time in transplanted tissue on an eye of thesubject, wherein the tissue is from an organ of interest, and whereinthe morphological changes over time in the transplanted tissue on theeye of the subject indicates a physiological status of the organ ofinterest in the subject.

As shown in the examples that follow, the inventors have demonstrated anovel methodology based on the use of a few “reporter islets”transplanted into the anterior chamber of the eye, serving as in vivoindicators of morphological changes occurring in situ in the pancreaticislet population over a course of several months. This concept isillustrated in the examples by the longitudinal visualization andquantification of obesity-induced islet growth and vascularizationpatterns that were subsequently normalized after drug treatment, showingfor the first time evidence for individual islet remodeling by theexpansion or reduction of its insulin-secretory potential in adaptationto specific needs. Hence “reporter islets” serve as optically accessibleindicators of islet function in the pancreas, and can be used aspersonalized in vivo biological markers serving as efficient readoutsfor both diagnosing islet malfunction and monitoring the effects ofspecific treatments on the regulation of islet cell mass and functionalstatus.

The subject can be any subject of interest, preferably a mammal,including but not limited to mice, rats, rabbits, dogs, cats, primates,chimps, baboons, and humans.

While the methods are exemplified by use of reporter islets as to assesspancreatic physiological status, it will be understood by those of skillin the art, based on the teachings herein, that the methods can beapplied to a wide range of organ types by use of transplanted tissuesfrom the organ of interest. The physiologic status of any suitable organof interest can be assessed by the methods of the invention. In oneembodiment, the organ of interest is selected from the group consistingof pancreas, lung, heart, brain, kidney, liver, small intestine, largeintestine, colon, stomach, gall bladder, esophagus, ureter, urethra,ovary, uterus, breast, spinal cord, prostate, hypothalamus, adrenalgland, pituitary gland, thyroid gland, parathryroid gland, pineal gland,spleen, thymus, rectum, sweat glands, salivary glands, mammary gland,seminal vesicles, glomeruli, tumor, fat tissue, and testes.

In another embodiment, the organ of interest comprises an exocrinegland. In this embodiment, non-limiting examples of exocrine glandsinclude sweat glands, salivary glands, mammary glands, stomach, liver,and pancreas. In a further embodiment, the organ of interest comprisesan endocrine gland. In this embodiment, non-limiting examples ofendocrine glands include pituitary gland, pancreas, ovaries, testes,thyroid gland, and adrenal gland.

The transplanted tissue may comprise individual cells, a plurality ofcells of the same type, or a plurality of different cell types, such astissues/tissue portions. The transplanted tissue may be obtained fromany suitable source. In one preferred embodiment, the transplantedtissue is obtained from the subject prior to transplantation in the eye.Methods for obtaining small amounts of tissue from organs of interestare well known to those of skill in the art.

Any suitable amount of tissue may be transplanted that minimizes anynegative impact of the transplant on vision or other eye function in thesubject. As exemplified in the data shown herein, 10-20 islets weretransplanted into the anterior chamber of the mouse eye. Based on theteachings herein, those of skill in the art can determine an appropriateamount of tissue to be transplanted for a given subject.

In one embodiment, monitoring of morphological changes in thetransplanted tissue begins approximately one month aftertransplantation, to permit full vascularization and innervation of thetransplanted tissue. In another embodiment, monitoring of morphologicalchanges in the transplanted tissue can begin as soon as desired aftertransplantation; this embodiment can be used, for example, when fullvascularization of the transplanted tissue is not required for a givenstudy (for example, when looking at test compound effects onvascularization and/or innervation). In all embodiments, the monitoringcan be carried on as long as desired for a given study. If desired, thetransplanted tissue may be removed after completion of treatment or forany other reason.

In one preferred embodiment, the organ of interest is a tumor. In thisembodiment, the methods may be used, for example, on a non-humanmammalian subject to test candidate compounds or other therapies foranti-tumor efficacy, side effects, etc.

In a further preferred embodiment, the organ of interest is thepancreas. In this embodiment, the tissue may be any suitable pancreatictissue, including but not limited to isolated pancreatic islets ofLangerhans. Islets of Langerhans are composed of several different celltypes, including alpha-, beta- and delta-cells. These clusters of cellsrepresent the endocrine pancreas and are of major importance for glucosehomeostasis. Insufficient release of insulin from beta-cells in responseto elevated blood glucose levels leads to diabetes. The regulation ofglucose induced insulin secretion from beta-cells is a complex process,modulated by autocrine, paracrine, hormonal and neuronal factors.

Any morphological changes can be assessed as suitable for a given study,and depend at least in part on the type of transplanted tissue and theorgan of interest. In non-limiting embodiments, the morphologicalchanges include, but are not limited to cell size in the tissue, cellvolume in the tissue, cell area in the tissue, cell shape in the tissue,cell death in the tissue, cell proliferation in the tissue, cell mass inthe tissue, blood perfusion in the tissue, optical reflectivity of cellsin the tissue, and granulation of cells in the tissue.

For example, in embodiments where the organ of interest is the pancreasand the transplanted tissue comprises isolated islets of Langerhans,exemplary morphological changes include:

(a) changes in beta cell mass (reflects pancreatic physiologic status);

(b) beta cell destruction (may reflect pancreatic pathology);

(c) beta cell hyperplasia and/or cellular hypertrophy (reflectsincreased insulin content)

(d) beta cell proliferation (reflects pancreatic physiologic status);

(e) islet dimensions (reflects pancreatic physiologic status);

(f) islet damage (may reflect pancreatic pathology);

(g) intra-islet blood vessel diameter (may reflect compensatorymechanism designed to increase microvascular blood perfusion underhyperglycemic conditions);

(h) islet degranulation (reflects insulin secretion levels); and

(i) islet optical reflectivity (indicative of insulin release).

In one embodiment of any embodiment or combination of embodiments of theinvention, the methods may be used to monitor the effect on the organ ofinterest of a therapeutic or test compound administered to the subject.In one non-limiting example, the methods comprise assessing efficacy ofa course of treatment for a disorder in the subject. In one non-limitingembodiment, the subject suffers from diabetes (type 1 or type 2diabetes), or the subject is an animal model of diabetes and the methodsare used to monitor efficacy of a course of treatment that the subjectis undergoing to treat the diabetes. In one embodiment, the course oftreatment comprises administration of a therapeutic or candidatetherapeutic to the subject, and the method monitors efficacy of thetherapeutic, side effects of the therapeutic, effects of dosage changes,and/or any other endpoint of interest in the individual. In anotherembodiment, the course of treatment comprises a diet and/or an exerciseregimen, and wherein the method monitors efficacy of the diet and/orexercise regimen in the individual.

As exemplified below, in vivo imaging of transplanted islets provides3-dimensional morphological information that allows for a precisequantification of various islet parameters at given time points aftertransplantation. For example, quantification of average islet volumesover time showed significant differences between ob/ob and controlsubjects starting from 1 month after transplantation, illustrating adramatic islet growth in the ob/ob subject. This growth proved to beindependent of the subject donor genotype, i.e. a similar growth wasobserved when transplanting control subject islets into ob/ob subject,demonstrating that i) signaling factors originating from the recipientsubject dictate morphological changes of transplanted islets, and ii)the transplanted islets reflect morphological behavior of therecipient's in situ pancreatic islets. The studies shown below furtherdemonstrate that this observed plasticity is reflective of theplasticity occurring in situ in islets located in the pancreas, and thatin vivo changes in these morphological parameters caused by treatmentfor the disorder were also found in the transplanted islets.

Thus, the examples demonstrate that “reporter islets” can reveal, in arepresentative way, the remodeling of the in situ pancreatic isletpopulation, expanding or reducing their insulin-secretory potential.Hence, this technique permits to “merge” the study of individual isletswith the study of islet populations, and has the potential to replacemultiple cross-sectional experiments with longitudinal studies. We thuspropose the in vivo imaging of “reporter” transplanted tissue into theeye as a versatile tool to identify factors leading to morphologicalchanges in the transplant, and thus the organ of interest.

Transplantation into the eye preferably involves transplantation intothe anterior chamber of the eye. The anterior chamber of the eyecomprises the front portion of the eye, and includes the structure infront of the vitreous humour, as well as the cornea, iris, ciliary body,and lens. Transplantation of the tissue into the anterior chamber of theeye can comprise placement of the cells into any one or more of theseanterior eye chamber compartments. In one non-limiting example, tissuetransplants are transplanted via injection through the cornea, allowingengraftment of the transplanted islets onto the iris, permittingobservation and imaging through the cornea. Islets transplanted into theanterior chamber of the eye engrafted on the iris, became vascularized,retained their cellular composition, and responded to stimulation.Furthermore, they can be monitored by non-invasive laser scanningmicroscopy (LSM) allowing in vivo imaging of morphological changes. See,for example, published US patent application 20090060843. Employing theanterior chamber of the eye as a transplantation site permits continuousmonitoring of morphological changes in the transplanted tissue, and canbe used to elucidate effects of modulatory inputs from, for example, thehormonal and neuronal system, as well as from autocrine/paracrinesignals of endocrine or vascular cells.

The morphological changes in the transplanted tissue can be monitored byany suitable technique, including but not limited to directvisualization. In a preferred embodiment, the morphological changes aremonitored by microscopy. See, for example, published US patentapplication 20090060843, and the methods disclosed in the examples thatfollow. In one preferred embodiment, the morphological changes aremonitored by confocal microscopy and/or two-photon microscopy, whichprovide 3-dimensional morphological information.

The methods comprise monitoring morphological changes over time intransplanted tissue on an eye of the subject. In one embodiment, thesubject is administered a therapeutic or test compound of interest viaany suitable route of administration, and morphological changes in thetransplanted tissue are determined at one or more (i.e.: 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more) subsequent time points. Any suitable amount oftime between measurements of morphological change can be employed, asdeemed appropriate in light of the specifics of the methods beingcarried out.

In another embodiment, the methods further comprise comparing themorphological changes over time in the transplanted tissue to changes inin situ changes occurring over the same time frame as represented inbiopsies obtained from the subject. The in situ changes can be assessedby any suitable technique, including but not limited to histochemicalanalysis of tissue sections as described in the examples that follow.

Example: Reporter Islets Reveal the Adaptive Plasticity of In SituPancreatic Islets

Abstract

The islets of Langerhans constitute the endocrine pancreas and areresponsible for maintenance of blood glucose homeostasis. They aredeeply embedded in the exocrine pancreas and therefore theiraccessibility for functional studies is limited. To understandregulation of function and survival and assess the clinical outcome ofindividual treatment strategies for diabetes, a monitoring systemcontinuously reporting on the endocrine pancreas is needed. We describethe application of a natural body window that successfully reports onthe properties of in situ pancreatic islets. As proof of principle“reporter islets” were transplanted into the anterior chamber of the eyeof leptin-deficient mice. The “reporter islets” displayedobesity-induced growth and vascularization patterns that were reversedby leptin treatment. Hence “reporter islets” serve as opticallyaccessible indicators of islet function in the pancreas, and reflect theefficiency of specific treatment regimens regulating islet plasticity invivo.

Introduction

Normal fluctuations in blood glucose concentration trigger anorchestrated release of hormones from various cells in the endocrinepancreas, the islets of Langerhans. The beta cell produces and secretesinsulin, an essential hormone regulating glucose uptake. A decrease infunctional beta cell mass leads to impaired glucose homeostasis anddiabetes, a devastating disease with epidemic spreading. An adequateregulation of beta cell mass is thus of paramount importance to adapt tovarious functional demands always ensuring maintenance of a normal bloodglucose concentration (1). The major challenge for functional studies ofthe islets of Langerhans in health and disease is the fact that they aredeeply embedded in the exocrine pancreas and therefore have a limitedaccessibility. Hence, to understand regulation of function and survivaland to assess the clinical outcome of individual treatment strategiesfor diabetes, there is an ultimate need for a monitoring systemcontinuously reporting on the status of the endocrine pancreas in theliving organism.

We have developed a technical platform for non-invasive, longitudinal,in vivo imaging at single cell resolution (2, 3). By applying this invivo imaging technique to the current study, we wanted to test ourhypothesis that islets transplanted into the anterior chamber of the eyecan report on the functional status of the endogenous endocrine pancreasand that intervention with pathological processes in the islets ofLangerhans indeed can be monitored in the eye. For this purpose we tookadvantage of the obese mutant mouse (ob/ob) as a model system. Thismouse was first described in 1950 (4), it displays impressive isletplasticity during its lifetime and has been extensively studied as amodel for obesity and insulin resistance. At a very young age these miceare hyperinsulinemic, hyperglycemic and show a higher than average bodyweight (5). In addition to a distinct obesity coupled to a strongappetite, these mice display a number of impaired functions, such asreduced metabolic rate, impaired thermogenesis, impaired immunity andinfertility (6). In 1994, Friedman and coworkers identified the ob geneencoding for the hormone leptin, produced mainly in adipose tissue (7).This gene is mutated in the ob/ob mouse and as a consequence this mouseis incapable of expressing any functional leptin. Under normalconditions leptin has many different physiological roles but one of itsmost remarkable functions is to regulate appetite. In response tonutrition, leptin is released from adipose tissue and activates leptinreceptors in the hypothalamus, leading to suppression of appetite and asa consequence reduced food intake. Additionally, leptin receptors areexpressed in beta cells and are involved in an adipo-insular feedbackloop inhibiting insulin expression and release after food intake (8, 9).The lack of leptin in the ob/ob mouse thus results in rapid increase inbody weight and, in an effort to compensate for the increased demand forinsulin, in beta cell hyperplasia (10). As a consequence the islet cellpopulation of the ob/ob mouse is altered, the percentage of beta cellsin comparison to other endocrine cells is particularly high and accountsfor more than 90% of total islet cells (11). In the ob/ob mouse theabsence of a leptin-driven feedback loop both explains the stronginsulin release observed from beta cells, resulting in apparentdegranulation (10) and the development of insulin resistance.

By applying the ob/ob mouse as a model system, we could demonstrate theversatile and interesting application of optically-accessible “reporterislets” in the anterior chamber of the eye. These reporter isletssuccessfully monitor beta cell plasticity in situ in the endocrinepancreas and also enable the follow up of a specific treatment regimen.

Results

The ob/ob Mouse Displays Abnormal Physiological Properties

The ob/ob mouse can be morphologically differentiated from its controllittermate already at the age of 4 weeks, and its increased body massbecomes increasingly noticeable with advancing age (FIG. 1A, B).Physiological studies revealed increased fasting blood glucose andinsulin levels as compared to control littermates (FIG. 1C). This is inaccordance with earlier reports (5) and is indicative of excessive foodintake and insulin resistance. Paraffin-embedded sections of ob/ob mouseand control mouse pancreata were stained by hematoxylin and eosin toobserve differences in islet morphology. Islet dimension was increasedin the ob/ob mouse to provide a large potential insulin secretorycapacity in an effort to compensate for the increased food intake (FIG.1D, E). While looking at pancreas sections provides informationregarding the morphological status of islets at specific time points,dynamic changes cannot be appreciated.

Intraocular Islet Transplants Mirror the Adaptive MorphologicalPlasticity of In Situ Endogenous Islets

To study the morphological plasticity of these islets over time wetherefore transplanted a few “reporter islets” into the anterior chamberof the mouse eye, which can be optically accessed for longitudinal invivo imaging. Pancreatic islets were isolated from donor mice at the ageof 4 weeks and transplanted into the anterior chamber of the eye ofage-matched recipients. This in vivo environment contains a richcapillary as well as nervous network that will connect to the engraftedislets and thus permit not only inter-cellular paracrine but alsoendocrine and nervous input (2, 12). Transplanted islets rapidlyengrafted onto the iris and individual islet transplants could beidentified repetitively at different time points after transplantation(FIG. 2A). In addition to an increased growth these islets displayedincreased intra-islet vessel diameters in the ob/ob recipient (FIG. 2B),as previously documented from dissected pancreatic tissue in vitro (13,14). The increase in intra-islet vessel diameter could be implicated ina compensatory mechanism designed to increase blood perfusion underhyperglycemic conditions (15). It might also be one approach to optimizeendocrine signaling, ensuring that each of the beta cells will have adirect communication with blood flow under this strong proliferativecondition. Individual islets were imaged in vivo at different timepoints in syngeneically transplanted mice by confocal microscopy, andthe large and tortuous blood vessels in the ob/ob islet grafts wereconfirmed by fluorescence imaging (FIG. 2C). Backscatter imagingrevealed islet morphology as well as a seemingly degranulated and unevenpattern in the ob/ob mouse islet. The scarce reflection of light isindicative of rapid insulin secretion, which can be explained bydisruption of the adipo-insular feedback loop under leptin-deficientconditions (9). Analysis of in situ islets by immunohistochemistry onparaffin-embedded sections showed that islets in the pancreas followsimilar distinctive properties between ob/ob and control mice, i.e. CD31staining showed large vessels in the ob/ob islets and insulin stainingwas irregular.

The in vivo imaging of transplanted islets by confocal microscopyprovides 3-dimensional morphological information that for the first timeallows for a precise quantification of islet volume at any given timepoint after transplantation. Quantification of average islet volumesover time showed significant differences between ob/ob and control micestarting from 1 month after transplantation, illustrating a dramaticislet growth in the ob/ob mouse (FIG. 2D, E). Interestingly, this growthproved to be independent of the mouse donor genotype, i.e. we observed asimilar growth when transplanting control mouse islets into ob/ob mice,demonstrating that in this particular case i) signaling factorsoriginating from the recipient mouse dictate morphological changes oftransplanted islets, and ii) the transplanted islets reflectmorphological behavior of the recipient's in situ pancreatic islets.

To examine whether this observed plasticity in the anterior chamber ofthe eye indeed reflects the plasticity occurring in situ in isletslocated in the pancreas, we compared paraffin-embedded sections of islettransplants and endogenous pancreatic islets by immunohistochemistry. Weobserved a strong proliferation of beta-cells in the ob/ob mouse isletsby staining for insulin and Ki67 (FIG. 2F). This proliferation rate wasnot significantly different whether measured from in situ pancreaticislets or from islets engrafted in the anterior chamber of the eye(mean±s.e.m.=0.82±0.10%, n=5, and 0.95±0.15%, n=4 mice, respectively).Morphological changes in beta cells were also assessed byimmunohistochemistry and revealed an approximately 1.35-fold increase incell area in the ob/ob mice as compared to control mice, both in in situislets located in the pancreas and in islet grafts (FIG. 2G). The lackof leptin signaling is a likely contributor to this volume expansion.Indeed it has been shown, using pancreas-specific leptin receptor KOmice, that the disruption of leptin action in islets results in enhancedPI3K/AKT signaling, associated with increases in beta cell size andislet mass (16). Although this compensatory islet growth has beendemonstrated in vitro, we have now been able to verify this pattern inthe living ob/ob mouse longitudinally by the analysis of single“reporter islets”. Importantly, our data are compatible with previousstudies where the mean islet volume showed a 4-fold difference betweencontrol and ob/ob mice at 2 months of age, either from studies based onisolated islets (17) or from imaging of histological sections spanningthe entire pancreas (18).

Leptin Treatment Reduces Body Weight and Corrects Blood GlucoseHomeostasis in the ob/ob Mouse

Having witnessed the behavior of individual islets in response to foodover-consumption and insulin resistance, we next questioned if thisgrowth could be refrained or even reversed by treating the ob/ob micewith leptin. Mice were injected intraperitoneally every day between 3and 4 months of age. The physiological effects of this treatment weremonitored regularly using different parameters. Parallel to theirvisibly reduced appetite we measured a decrease in body weight, bloodglucose and insulin concentrations during treatment (FIG. 3A). However,this beneficial effect of leptin was not permanent as body weightrapidly increased after the end of the treatment period, to reach a masssimilar to age-matched untreated ob/ob mice about one month later (seeFIG. 1B for comparison). The treatment also had a beneficial impact oninsulin sensitivity. At the end of the treatment period, intraperitonealglucose tolerance tests on ob/ob mice demonstrated glucose excursionssimilar to those obtained from age-matched control mice (FIG. 3B).

Longitudinal in vivo imaging reveals the reversed dysregulation of ob/obmouse islets by leptin

The leptin treatment also exerts an influence on islet growth. Whereassham-treated mice displayed a continuous adaptive growth of islettransplants, this growth was abolished and partially reversed underleptin-treatment (FIG. 4A, B), in accordance with earlier reports (19,20). At the end of the treatment the proliferation of beta cells wasvirtually abolished in both the engrafted islets and the in situpancreatic islets from leptin-treated ob/ob mice (FIG. 4C). Followingleptin treatment the average beta cell size reverted and showed nosignificant difference compared to age-matched untreated control mice,independently of whether the islets were transplanted or endogenouslypresent in the pancreas (data not shown). The decrease in islet sizecould be explained in part by this decrease in individual beta cellsize, as previously reported (21) and by morphological changes inintra-islet vasculature (FIG. 5). We measured a decrease in vesseldiameter in islet grafts that was parallel to the lowered blood glucoselevels. Interestingly, we could observe angiogenesis in the islet graftsat an early stage after leptin treatment, a biological phenomenon knownto enhance blood perfusion and to be positively influenced by leptin viaa synergistic stimulation with the angiogenic factors FGF-2 and VEGF(22).

Because of the restored leptin-driven adipo-insular feedback loop andthe decrease in blood glucose levels we could observe the resultingregranulation of islet cells in vivo in transplanted islets by theirstronger reflective optical properties (FIG. 4A), in accordance with theincreased immunostaining for insulin perceived in islets fromleptin-treated ob/ob mice (19). The normalization of insulin release invivo after leptin treatment in the ob/ob mouse has been shown to be theresult of mechanisms both dependent and independent on food intake (20).

Discussion

To test our novel concept of “reporter islets” reporting on the statusof the in situ endocrine pancreas, we took the advantage of the factthat there is a significant remodeling capacity of the endocrinepancreas in the leptin-deficient ob/ob mouse. “Reporter islets” from theob/ob mouse model were thus transplanted into the anterior chamber ofthe eye of a similar type of mouse for the investigation of islet cellmass regulation, representative of that going on in the endocrinepancreas. The “reporter islets” displayed obesity-induced growth andvascularization patterns that were identical to those observed in situin the pancreas. These obesity-induced growth and vascularizationpatterns were reversed by leptin treatment both in the anterior chamberof the eye and in the in situ pancreas, showing evidence for a highlyeffective islet remodeling by the expansion or reduction of its insulinsecretory potential. Such a remodeling capacity has so far only beenindirectly suggested by cross-sectional studies or by the analysis ofpancreatic islet populations at relatively low resolution in vitro.Hence, “reporter islets” can successfully reveal the molecularmechanisms regulating adaptive plasticity of the endocrine pancreas invivo.

A number of different experimental approaches allow for thequantification of beta cell mass and for eventually displaying theremodeling of islets. In vitro techniques include the analysis ofhistological sections of pancreas by point counting morphometry (18) andthe direct measurements of islet dimensions in dissected pancreatictissue (23, 24). Ex vivo imaging techniques comprise the imaging ofexteriorized pancreas by confocal microscopy (25) or optical coherencemicroscopy (26). Finally, several noninvasive in vivo imaging techniquesaim at the longitudinal quantification of total beta cell mass, i.e.magnetic resonance imaging (MRI (27)), positron emission tomography (PET(28)), bioluminescence imaging (29), or combined multimodal imaging(30). None of these techniques offer the possibility to followmorphological changes in individual islets over time.

We now show that a few “reporter islets” can reveal, in a representativeway, the remodeling of the in situ pancreatic islet population,expanding or reducing their insulin-secretory potential. Hence, thistechnique permits to “merge” the study of individual islets with thestudy of islet populations, and has the potential to replace multiplecross-sectional experiments with longitudinal studies. We thus proposein vivo imaging of “reporter islets” transplanted into the anteriorchamber of the eye as a versatile tool to clarify molecular mechanismsas well as identify pharmacological compounds in the regulation of betacell function and survival. Importantly, in humans a homologous use of“reporter islets”, to both diagnose islet malfunction and monitoreffects of specific individually based treatment regimens, may beenvisaged as a novel personalized medicine approach. Our concept of“reporter islets” can be further developed by implementation of multiplehigh temporal cellular biomarkers for function and proliferation andalso extended to other organs, with the ambition of identifying andunderstanding in detail molecular interactions and adaptive mechanismsat the cell and organ level, having implications for both physiology andpathology.

Methods Summary

Transgenic Mice Models

The ob/ob mice used for our experiments originate from Umeå, Sweden, andare inbred in our animal core facilities at the Karolinska Hospital. Allexperiments were performed according to Karolinska Institutet'sguidelines for the care and use of animals in research and approved bythe local animal ethics committees at Karolinska Institutet.

Transplantation of Pancreatic Islets into the Anterior Chamber of theEye

Islets were isolated from female donor mice and transplanted into theanterior chamber of the eye of male recipients. We anesthetized miceusing isoflurane (Baxter), and approximately 10-20 islets weretransplanted into the anterior chamber of the mouse eye. Islets used fortransplantation were selected to be of similar size for each experiment,independent of the mouse genotype. The mice were injected subcutaneouslywith Temgesic (Schering-Plough) for post-operative analgesia.

In Vivo Imaging of Intraocular Islet Grafts

Islet grafts in mice were imaged in vivo at specific time points aftertransplantation. We used an upright laser scanning confocal microscopebased on TCS-SP2-AOBS (Leica) with a long-distance water-dippingobjective (Leica HXC-APO 10×/0.30NA), and a custom-built stereotaxichead holder allowing to position the mouse eye containing the engraftedislets towards the objective. Viscotears (Novartis) was used asimmersion liquid between the eye and the objective, and isoflurane wasused to anesthetize the mice during in vivo imaging. Imaging of isletmorphology was done by laser illumination at 633 nm, and collection ofbackscattered light at the same wavelength.

Leptin Treatment

Leptin treatment was performed by daily intraperitoneal injections of1.5 μg/g body weight of recombinant human leptin (AmylinPharmaceuticals), and sham treatment was performed by intraperitonealinjection of water instead of leptin.

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(2001) Long-term leptin treatment of ob/ob mice    improves glucose-induced insulin secretion. Int J Obes Relat Metab    Disord 25:816-821.-   20. Lee J-W, Romsos D R (2003) Leptin administration normalizes    insulin secretion from islets of Lep(ob)/Lep(ob) mice by food    intake-dependent and -independent mechanisms. Exp Biol Med (Maywood)    228:183-187.-   21. Park S, Hong S M, Sung S R, Jung H K (2008) Long-term effects of    central leptin and resistin on body weight, insulin resistance, and    beta-cell function and mass by the modulation of hypothalamic leptin    and insulin signaling. Endocrinology 149:445-454.-   22. Cao R, Brakenhielm E, Wahlestedt C, Thyberg J, Cao Y (2001)    Leptin induces vascular permeability and synergistically stimulates    angiogenesis with FGF-2 and VEGF. Proc Natl Acad Sci USA    98:6390-6395.-   23. Parsons J A, Bartke A, Sorenson R L (1995) Number and size of    islets of Langerhans in pregnant, human growth hormone-expressing    transgenic, and pituitary dwarf mice: effect of lactogenic hormones.    Endocrinology 136:2013-2021.-   24. Alanentalo T et al. (2008) High-resolution three-dimensional    imaging of islet-infiltrate interactions based on optical projection    tomography assessments of the intact adult mouse pancreas. J Biomed    Opt 13:054070.-   25. Nyman L, Ford E, Powers A, Piston D (2010) Glucose-dependent    blood flow dynamics in murine pancreatic islets in vivo. Am J    Physiol Endocrinol Metab 298:E807-14.-   26. Villiger M et al. (2009) In vivo imaging of murine endocrine    islets of Langerhans with extended-focus optical coherence    microscopy. Diabetologia 52:1599-1607.-   27. Lamprianou S et al. (2011) High-resolution magnetic resonance    imaging quantitatively detects individual pancreatic islets.    Diabetes 60:2853-2860.-   28. Singhal T et al. (2011) Pancreatic beta cell mass PET imaging    and quantification with [11C]DTBZ and [18F]FP-(+)-DTBZ in rodent    models of diabetes. Mol Imaging Biol 13:973-984.-   29. Park S-Y, Bell G I (2009) Noninvasive monitoring of changes in    pancreatic beta-cell mass by bioluminescent imaging in MIP-luc    transgenic mice. Horm Metab Res 41:1-4.-   30. Virostko J et al. (2011) Multimodal image coregistration and    inducible selective cell ablation to evaluate imaging ligands.    Proceedings of the National Academy of Sciences 108:20719-20724.-   31. Ellett J, Evans Z, Zhang G, Chavin K, Spyropoulos D (2009) A    rapid PCR-based method for the identification of ob mutant mice.    Obesity (Silver Spring) 17:402-404.    Materials and Methods    Mouse Model

The ob/ob mice used for our experiments originate from Umeå, Sweden, andare inbred in our animal core facilities at the Karolinska Hospital. Thediscrimination from control lean littermates was achieved by phenotypicand genotypic analysis (31). All experiments were performed according toKarolinska Institutet's guidelines for the care and use of animals inresearch and approved by the local animal ethics committees atKarolinska Institutet.

Physiological Measurements and Leptin Treatment

Body weight and blood glucose levels were measured in a minimum of 7mice. Glucose concentrations were obtained using Accu-Chek™ Avivamonitoring system (Roche). Glucose tolerance tests were performed byintraperitoneal injection of 2 mg glucose per gram body weight in micefasted for 16 hours (n≥5). Plasma insulin concentrations from a minimumof 3 mice per condition were measured using mouse insulin ELISA plates(Mercodia). Leptin treatment was performed by daily intraperitonealinjections of 1.5 μg/g body weight of recombinant human leptin (AmylinPharmaceuticals), and sham treatment was performed by intraperitonealinjection of water instead of leptin.

Transplantation of Pancreatic Islets into the Anterior Chamber of theEye

Islets were isolated from female donor mice and transplanted into theanterior chamber of the eye of male recipients, using a previouslydescribed technique (2). We anesthetized mice using isoflurane (Baxter),and approximately 10-20 islets were transplanted into the anteriorchamber of the mouse eye. Islets used for transplantation were selectedto be of similar size for each experiment, independent of the mousegenotype. The mice were injected subcutaneously with Temgesic™(Schering-Plough) for postoperative analgesia.

In Vivo Imaging of Intraocular Islet Grafts

Islet grafts in mice were imaged in vivo at specific time points aftertransplantation as previously described (2). We used an upright laserscanning confocal microscope based on TCS-SP2-AOBS (Leica) with along-distance water-dipping objective (Leica HXC-APO 10×/0.30NA), and acustom-built stereotaxic head holder allowing to position the mouse eyecontaining the engrafted islets towards the objective. Viscotears™(Novartis) was used as immersion liquid between the eye and theobjective, and isoflurane was used to anesthetize the mice during invivo imaging. Imaging of islet morphology was done by laser illuminationat 633 nm, and collection of backscattered light at the same wavelength.For visualization of blood vessels we injected 100 μl of a solutioncontaining 2.5 mg/ml of 500 kDa FITC-labeled dextran (Invitrogen) intothe tail vein, and imaged fluorescence using 496 nm excitationwavelength. Scanning speed and laser intensities were adjusted to avoidany cellular damage to the mouse eye or islet graft.

Tissue Sections and Immunohistochemistry

Dissected tissues were briefly rinsed with PBS, fixed for 48 h at roomtemperature using formalin, and dehydrated and embedded in paraffin.Five-micron thick sections mounted on precoated microscope slides weredewaxed by xylene and progressively rehydrated before processing.Insulin was stained using chicken anti-insulin (1:200 dilution, Abcam)followed by goat anti-chicken Alexa™ 488 (1:1000 dilution, Invitrogen)antibodies; blood vessels were stained using rat anti-mouse CD31 (1:50dilution, BD Pharmingen), followed by incubation of biotinylated goatanti-rat (5 μg/ml, Vector Labs) and amplification withHRP-streptavidin/Alexa™ 647-tyramide (Invitrogen); proliferation wasassessed by using mouse anti-human Ki67 (1:50 dilution, Novocastra)together with M.O.M. biotinylated anti-mouse IgG (Vector Labs), andamplified using HRP-streptavidin/Alexa 647-tyramide. Nuclei were stainedand slides were preserved by cover glass mounting using ProLong™ GoldAntifade Reagent with DAPI (Invitrogen). Slides were imaged using a BDPathway 855 system (BD Biosciences). Insulin, CD31, Ki67 and DAPIstainings were imaged using a 20×/0.75NA UApo/340 Olympus objective, andoverviews of pancreatic sections stained by hematoxylin/eosin wereobtained by montage capture mode using a 4×/0.16NA UPlan SApo Olympusobjective.

Image Processing and Analysis

AutoQuant X2 (Media Cybernetics) was used for blind deconvolution of allin vivo confocal images before image analysis. Islet volume was analyzedbased on the backscatter signal channel, using Matlab™ and the ImageProcessing Toolbox (Mathworks). The islet “equatorial volume”, i.e. thevolume from the top of the islet down to the calculated equator, wasused to represent the volume and calculate islet growth. We analyzed aminimum of 3 islet grafts per animal to determine islet growth, with aminimum of 3 mice per category. Individual vessel segments wereidentified at different imaging time points and their diameter measuredusing Volocity™ (Perkin Elmer). We established image analysis protocolsin Volocity™ for automated analysis of histological sections. Theaverage beta cell section area was obtained by dividing theinsulin-stained area by the total number of DAPI-stained nuclei enclosedin this area (minimum of 1000 cells per tissue). Beta cell proliferationrate was obtained by counting Ki67-positive nuclei and dividing by thetotal number of DAPI-stained nuclei in insulin-positive cells (minimumof 10000 cells per transplanted eye or pancreas). Volocity™ was used forimage display and Photoshop CS5 (Adobe) for image assembly.

Statistical Analysis

All results are presented as average±SEM. Student's t test was used fordetermining statistical significance, and P values<0.05 were consideredsignificant.

I claim:
 1. A method for monitoring status of in situ pancreatic isletsin a human subject that has a disease relating to the pancreas,comprising monitoring changes over time in pancreatic islets ofLangerhans transplanted on an eye of the subject, wherein the changesover time in the transplanted pancreatic islets of Langerhans indicatestatus of in situ pancreatic islets in the subject.
 2. The method ofclaim 1, wherein the method is used to monitor efficacy of a course oftreatment for a disorder in the subject, wherein the changes over timein the transplanted pancreatic islets of Langerhans on the eye of thesubject occur in response to the treatment.
 3. The method of claim 2,wherein the course of treatment comprises administration of atherapeutic to the subject, and wherein the method monitors efficacy ofthe therapeutic in the subject.
 4. The method of claim 2, wherein thecourse of treatment comprises a diet and/or an exercise regimen, andwherein the method monitors efficacy of the diet and/or exercise regimenin the subject.
 5. The method of claim 1, wherein the transplantedpancreatic islets of Langerhans are obtained from the subject.
 6. Themethod of claim 2, wherein the transplanted pancreatic islets ofLangerhans are obtained from the subject.
 7. The method of claim 1,wherein the method further comprise administering a therapeutic to thesubject, and monitoring changes resulting from the administering.
 8. Themethod of claim 7, wherein the changes in the transplanted pancreaticislets of Langerhans are determined at two or more time points after theadministering.
 9. The method of claim 5, wherein the method furthercomprises administering a therapeutic to the subject, and monitoringchanges resulting from the administering.
 10. The method of claim 9,wherein the changes in the transplanted pancreatic islets of Langerhansare determined at two or more time points after the administering. 11.The method of claim 1, wherein the changes are monitored by microscopy.12. The method of claim 1, wherein the transplanted pancreatic islets ofLangerhans are present in an anterior chamber of the eye of the subject.13. The method of claim 1, wherein the transplanted pancreatic islets ofLangerhans are present on an iris of the eye of the subject.
 14. Themethod of claim 2, wherein the transplanted pancreatic islets ofLangerhans are present on an iris of the eye of the subject.
 15. Themethod of claim 5, wherein the transplanted pancreatic islets ofLangerhans are present on an iris of the eye of the subject.
 16. Themethod of claim 7, wherein the transplanted pancreatic islets ofLangerhans are present on an iris of the eye of the subject.
 17. Themethod of claim 1, wherein the disease relating to the pancreascomprises diabetes.
 18. The method of claim 2, wherein the diseaserelating to the pancreas comprises diabetes.
 19. The method of claim 5,wherein the disease relating to the pancreas comprises diabetes.
 20. Themethod of claim 13, wherein the disease relating to the pancreascomprises diabetes.
 21. The method of claim 1, wherein (a) thetransplanted pancreatic islets of Langerhans are obtained from thesubject; (b) the transplanted pancreatic islets of Langerhans arepresent on an iris of the eye of the subject; (c) the disease relatingto the pancreas comprises diabetes; and (d) the changes are monitored bymicroscopy.